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A Mesophilic Aeromonas salmonicida Strain Isolatedfrom an Unsuspected Host, the Migratory BirdPied Avocet

Antony T. Vincent 1, Alex Bernatchez 2,3,4, Joachim Frey 5 and Steve J. Charette 2,3,4,*1 INRS-Institut Armand-Frappier, Bacterial Symbionts Evolution, Laval City, QC H7V 1B7, Canada;

antony.vincent@iaf.inrs.ca2 Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval,

Quebec City, QC G1V 0A6, Canada; abernatchez1@outlook.com3 Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, Quebec City,

QC G1V 4G5, Canada4 Département de Biochimie, de Microbiologie et de Bio-Informatique, Faculté des Sciences et de Génie,

Université Laval, Quebec City, QC G1V 0A6, Canada5 Vetsuisse Faculty, University of Bern, CH-3001 Bern, Switzerland; joachim.frey@vetsuisse.unibe.ch* Correspondence: steve.charette@bcm.ulaval.ca; Tel.: 1-418-656-2131 (ext. 406914)

Received: 22 August 2019; Accepted: 15 November 2019; Published: 20 November 2019��

Abstract: Aeromonas salmonicida is a Gram-negative bacterium, known as a fish pathogen since itsdiscovery. Although the species was initially considered psychrophilic, a mesophilic subspecies(pectinolytica) and many other mesophilic strains still not attributed to subspecies have been describedin the last two decades. These mesophilic strains were sampled from various sources, includinghumans, and some of them are known to be pathogenic. In this study, we describe a strain, JF2480,which was isolated from the spleen, and also found the kidney and liver of a dead pied avocet(Recurvirostra avosetta), a type of migratory bird inhabiting aquatic environments. A core genomephylogenomic analysis suggests that JF2480 is taxonomically distant from other known A. salmonicidasubspecies. The genome sequence confirms that the strain possesses key virulence genes that arepresent in the typical A. salmonicida psychrophilic subspecies, with the exception of the genes encodingthe type three secretion system (T3SS). Bacterial virulence assays conducted on the surrogate hostDictyostelium discoideum amoeba confirmed that the strain is virulent despite the lack of T3SS. Bacterialgrowth curves showed that strain JF2480 grow well at 40 ◦C, the body temperature of the pied avocet,and even faster at 41 ◦C, compared to other mesophilic strains. Discovery of this strain furtherdemonstrates the extent of the phylogenomic tree of this species. This study also suggests thatA. salmonicida can infect a wider array of hosts than previously suspected and that we need to rethinkthe way we perceive A. salmonicida’s natural environment.

Keywords: Aeromonas salmonicida; mesophile; bird; pied avocet; Dictyostelium discoideum; pathogenesis

1. Introduction

Aeromonas salmonicida is a Gram-negative bacterium, which is ubiquitous in aquatic environments.This species is divided into five officially recognized subspecies: salmonicida, smithia, achromogenes,masoucida, and pectinolytica [1]. Strains of the subspecies salmonicida, smithia, achromogenes and masoucidacan infect a wide range of fish and are psychrophilic with their growth being limited to temperaturesnot higher than about 25 ◦C [1–3]. In 2000, the pectinolytica subspecies was isolated directly from apolluted river (Matanza River, Argentina) without having any known host [4]. This subspecies growsat 37 ◦C and is thus considered mesophilic. Some studies had previously reported the existence of

Microorganisms 2019, 7, 592; doi:10.3390/microorganisms7120592 www.mdpi.com/journal/microorganisms

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mesophilic A. salmonicida strains, known as the hybridization group 3 [HG3], isolated from humanand animal hosts [5–8]. However, the classification of these strains was made prior to the advent ofDNA sequencing and we now know that biochemical approaches, compared to molecular ones, cansometimes lead to misclassifications for A. salmonicida [9]. Consequently, the finding of the subspeciespectinolytica was a major turning point for the taxonomy of A. salmonicida and suggested a greaterdiversity among the species of this bacterium.

Many new mesophilic A. salmonicida strains have been discovered and described especially inrecent years. This was the case with the characterization of four Indian mesophilic A. salmonicida strains(Y47, Y567, Y577, A527) from undetermined subspecies and isolated from contaminated food [10–12].Mesophilic A. salmonicida strains with human clinical background were also reported in Europe andIndia, including from a 68-year-old diabetic woman on continuous ambulatory peritoneal dialysiswith abdominal pain and cloudy peritoneal fluid, in the blood of a 34-year-old female patient, fromthe skin infection of a 67-year-old immunocompetent male, in the right eye of a 55-year-old femalewho had recovered from cataract surgery, from a 15-year-old boy who had recovered from a fingersurgery, from the feces of a child suffering from acute gastroenteritis, and finally from a person havingcellulitis in a foot following trauma [13–18]. However, the pathogenicity of the isolates from humancases was not clearly demonstrated until a very recent study clearly showed the capacity of mesophilicA. salmonicida strains to infect mammals by doing infection experiments on mice [18].

The most recent phylogenomic analyses based on core genome revealed a great diversity in themesophilic strains of the A. salmonicida species, opening the door to potentially more mesophilicsubspecies than psychrophilic ones [11,18]. The increasing number of mesophilic strains also suggeststhe possibility to find other mesophilic A. salmonicida subspecies in unsuspected hosts.

In this study, we investigated the first case of bird infection by a mesophilic A. salmonicida strain.The strain was isolated from the spleen, and also found in the kidney and liver of a pied avocet(Recurvirostra avosetta) dead of sepsis. Pied avocets are migratory birds that feed in shallow lakes andmud ponds. The complete genome of this strain was sequenced and its growth capacity, virulence,and metabolic properties were investigated. This strain confirms the evolution of A. salmonicida speciesto infect three different classes of hosts (fish, mammals, and birds).

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

A. salmonicida strains used in this study are described in Table 1. The strain that was retained andkept as JF2480 was an isolated colony from the spleen culture of a dead pied avocet. The spleen, liver,and kidney isolates were verified as A. salmonicida by phenotypic tests and sequencing rrs (16S rRNA),gyrA, and rpoB genes, but the latter two were not kept as they were identical to JF2480. When requiredfor the experiments, the bacteria were thawed from glycerol 15% stock and grown on tryptic soy agar(TSA) for one day at 37 ◦C for the mesophilic strains, or three days at 18 ◦C for the masoucida subspecies.

Table 1. A. salmonicida strains used in this study.

Name Subspecies Lifestyle Origin Reference

JF2480 N/A Mesophile Switzerland This studyY47 N/A Mesophile India [10]Y567 N/A Mesophile India [10]Y577 N/A Mesophile India [10]A527 N/A Mesophile India [11]

34 mel T pectinolytica Mesophile Argentina [4]NBRC 13784 T masoucida Psychrophile Japan [19]

T: type strain.

2.2. DNA Extraction and Sequencing

The total genomic DNA of strain JF2480 was extracted using DNeasy Blood and Tissue kits (Qiagen,Montreal, QC, Canada) with the addition of a RNase treatment step to the manufacturer’s protocol.Sequencing was performed using a MiSeq (Illumina, San Diego, CA, USA) system at the Plateformed’Analyse Génomique of the Institut de Biologie Intégrative et des Systèmes (Université Laval,Quebec City, QC, Canada).

2.3. Sequence Assembly and Analyses

The sequencing reads were de novo assembled using A5-miseq version 20160825 [20]. The draftgenome was annotated by Prokaryotic Genome Annotation Pipeline (PGAP) [21] and depositedin DDBJ/ENA/GenBank under the accession number VOIP00000000. The sequencing reads weredeposited in SRA under the accession number PRJNA264317.

Molecular phylogeny was used to determine the phylogenetic position of strain JF2480 among theother A. salmonicida. To do this, the genomic sequence of strain JF2480 was added to a dataset comprisinga representative of each of the Aeromonas species, all sequences from mesophilic A. salmonicida andthe sequences of representatives of all psychrophilic subspecies of A. salmonicida (Table S1) [18].All the 55 genome sequences were annotated using Prokka version 1.13.3 [22]. Homologous linksbetween the translated coding sequences were found using the combination of the two algorithmsCOG [23] and OMCL [24] through GET_HOMOLOGUES version 20190102 [25]. The 2018 genesequences (excluding paralogs) corresponding to the softcore (sequences present in more than 95% ofthe genomes) were aligned by codons using mafft version 7.407 [26] through TranslatorX version 1.1 [27].The resulting alignments were filtered using BMGE version 1.12 [28] and concatenated in a partitionedsupermatrix using AMAS [29]. The evaluation of the best-fit model of each partition (Table S2) andthe maximum-likelihood phylogeny were done using IQ-TREE version 1.6.10 [30,31]. The robustnessof the tree was assessed by performing 10,000 ultrafast bootstraps [32]. The final tree has beenmidpoint rooted using FigTree version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). The AverageNucleotide Identity (ANI) values were computed for genome sequences of A. salmonicida usingpyani (https://github.com/widdowquinn/pyani). The percentage of conserved proteins (POCP) werecomputed for each pair of genomes using the OMCL algorithm [24] through GET_HOMOLOGUESversion 20190102 [25]. Genes unique to JF2480 were found using PATRIC [33] and those involved insecretion systems with TXSScan [34]. EggNOG-mapper was used to further annotate the genes [35].

2.4. Virulence Assay

The virulence of A. salmonicida can be determined by its capacity to resist to the predation ofDictyostelium discoideum amoebae [36]. The strain DH1−10 of D. discoideum was grown in a Petri dishat 21 ◦C in HL5 liquid medium containing 15 µg/mL of tetracycline [37,38]. The D. discoideum cellswere harvested, centrifuged, resuspended in HL5 without antibiotic, at the following concentrations:100,000 cells/5 µL, 10,000 cells/5 µL, 1000 cells/5 µL, 100 cells/5 µL, 10 cells/5 µL and 0 cells/5 µL. Singlecolonies from A. salmonicida strains and from Klebsiella aerogenes strain, used as non-pathogenic control,were resuspended in 300 µL of tryptic soy broth (TSB) to an optical density of 0.4 at 595 nm (OD595)and 50 µL of the suspension were deposited in the wells of a 24-well plate containing 2 mL of HL5 agar.The dried bacterial lawns were spotted with 5 µL drops of D. discoideum cell suspension. The plateswere incubated for 7 days at 18 ◦C and were then examined for the presence of phagocytic plaques [36].

2.5. Growth Curve

Bacteria from overnight growth on TSA at 37 ◦C (for all bacteria except masoucida subspeciesgrown at 18 ◦C) were resuspended in TSB. The turbidity was adjusted to an OD595 of 0.2, and thecultures were incubated in 48-well plate at 40 ◦C or 41 ◦C with shaking at 200 rpm in a Tecan InfiniteF200 PRO microplate reader (Tecan, Morrisville, NC, USA). The ODs were measured automatically

every 15 min for 12 h. Statistical significance between the growth curves was assessed using thecompareGrowthCurves function of the R package statmod [39]. The p-values were adjusted for multipletesting using Holm’s method as implemented in compareGrowthCurves

3. Results

An Aeromonas sp. isolate initially identified as Aeromonas hydrophila-like strain was isolated in1998 from a dead pied avocet (Recurvirostra avosetta) (Figure 1) that lived in a pied avocet colony in anoutdoor enclosure of the zoo of Bern (Switzerland). The bird was the only dead one found in the colonyof which the other individuals showed no signs of disease. It was removed rapidly after death and hadundergone standard necropsy by a veterinary pathologist at the Department of Infectious Diseasesand Pathobiology, approximately 3 h after death. Other than a grossly enlarged spleen, the tissueswere reported free of macro-parasites and other lesions. Bacterial analysis was made immediatelyafter the necropsy. The Aeromonas sp. was isolated abundantly as virtually pure culture from thespleen, kidney, and liver shortly after the death of the animal, indicating its role as causative agent ofsepticemia and death of the bird. An isolate from the spleen named JF2480, was conserved and furthercharacterized based on rrs (16S rRNA) and rpoB sequence, which revealed that this bacterium was amember of the A. salmonicida species (data not shown). This was surprising since, to our knowledge, nocase of bird infection by A. salmonicida has been reported so far. It was even more intriguing knowingthat the body temperature of Recurvirostra avosetta is 40 ◦C [40], a relatively high temperature for thisbacterial species.

Microorganisms 2019, 7, x FOR PEER REVIEW 4 of 13

compareGrowthCurves function of the R package statmod [39]. The p-values were adjusted for

multiple testing using Holm’s method as implemented in compareGrowthCurves

3. Results

An Aeromonas sp. isolate initially identified as Aeromonas hydrophila-like strain was isolated in

1998 from a dead pied avocet (Recurvirostra avosetta) (Figure 1) that lived in a pied avocet colony in

an outdoor enclosure of the zoo of Bern (Switzerland). The bird was the only dead one found in the

colony of which the other individuals showed no signs of disease. It was removed rapidly after death

and had undergone standard necropsy by a veterinary pathologist at the Department of Infectious

Diseases and Pathobiology, approximately 3 h after death. Other than a grossly enlarged spleen, the

tissues were reported free of macro-parasites and other lesions. Bacterial analysis was made

immediately after the necropsy. The Aeromonas sp. was isolated abundantly as virtually pure culture

from the spleen, kidney, and liver shortly after the death of the animal, indicating its role as causative

agent of septicemia and death of the bird. An isolate from the spleen named JF2480, was conserved

and further characterized based on rrs (16S rRNA) and rpoB sequence, which revealed that this

bacterium was a member of the A. salmonicida species (data not shown). This was surprising since, to

our knowledge, no case of bird infection by A. salmonicida has been reported so far. It was even more

intriguing knowing that the body temperature of Recurvirostra avosetta is 40 °C [40], a relatively high

temperature for this bacterial species.

Figure 1. Pied avocets.

In order to have a clearer portrait of the identity of this bacterium, its genome was sequenced

using the Illumina MiSeq technology. A robust phylogenomic analysis based on core genome was

then performed to shed light on the phylogenetic relation between this unusual strain and the other

A. salmonicida strains (Figure 2). Interestingly, its phylogenetic position is between the previously

known mesophilic and psychrophilic strains. The ANI values confirm that it is, however, as distant

as the previously known mesophilic strains, while all the psychrophilic strains are more closely

related. To complement the ANI values, which permit us to measure the genetic distance in terms of

nucleotide identity, the POCP values were also calculated. The POCP values, that reflect the level of

conservation between different gene repertoires, clearly show that JF2480 is related to all other

mesophilic strains. In addition to clustering all mesophilic strains together, the POCP values permit

us to delineate the subspecies boundaries between psychrophilic subspecies [41]. Indeed, the

different psychrophilic subspecies share ~ 99% nucleotide identity between them, making their

Figure 1. Pied avocets.

In order to have a clearer portrait of the identity of this bacterium, its genome was sequencedusing the Illumina MiSeq technology. A robust phylogenomic analysis based on core genome wasthen performed to shed light on the phylogenetic relation between this unusual strain and the otherA. salmonicida strains (Figure 2). Interestingly, its phylogenetic position is between the previouslyknown mesophilic and psychrophilic strains. The ANI values confirm that it is, however, as distant asthe previously known mesophilic strains, while all the psychrophilic strains are more closely related.To complement the ANI values, which permit us to measure the genetic distance in terms of nucleotideidentity, the POCP values were also calculated. The POCP values, that reflect the level of conservationbetween different gene repertoires, clearly show that JF2480 is related to all other mesophilic strains.

In addition to clustering all mesophilic strains together, the POCP values permit us to delineate thesubspecies boundaries between psychrophilic subspecies [41]. Indeed, the different psychrophilicsubspecies share ~99% nucleotide identity between them, making their separation difficult basedon this criterion alone. However, POCPs have values around 80%, giving higher resolution thanANIs. This suggests a faster evolution of the gene repertoire in these subspecies compared to sequenceevolution. This result is corroborated by various studies showing a high plasticity in the mobilomeof A. salmonicida subsp. salmonicida and that this bacterium can easily acquire exogenous material byhorizontal transfers [42–44].

separation difficult based on this criterion alone. However, POCPs have values around 80%, giving

higher resolution than ANIs. This suggests a faster evolution of the gene repertoire in these

subspecies compared to sequence evolution. This result is corroborated by various studies showing

a high plasticity in the mobilome of A. salmonicida subsp. salmonicida and that this bacterium can easily

acquire exogenous material by horizontal transfers [42–44].

Figure 2. Cladogram showing the phylogenetic relations between JF2480 and other A. salmonicida

strains. Only bootstrap values inferior to 100 are shown at the corresponding nodes. The heatmap

represents the ANI values (in the middle) and the POCP values (on the right).

Given the unusual host, the genome sequence was an opportunity to investigate the virulence

factors of the strain JF2480. Secretion systems are protein machineries important for bacterial

virulence [45]. The genome of strain JF2480 was predicted to possess mandatory genes for T1SS, T2SS,

T4P, Tad, and T6SSi. Although no core genes encoding for T3SS have been detected, two genes coding

for effectors (AexT and AexU), known to be secreted by this system, were found.

In the past, it has been shown that bacteria without a functional T3SS and especially Aeromonas

bacteria were not virulent when tested in the predation assay involving the soil amoeba D. discoideum

used as a surrogate host [36,46]. A low quantity of amoebae was able to create a phagocytic plaque

on K. aerogenes (the positive control) while only a high quantity of D. discoideum cells was required to

observe amoebal growth on JF2480 (Figure 3). Thus, bacterial virulence assays conducted on JF2480

revealed that this strain is virulent when facing D. discoideum even in absence of a complete T3SS

compared to K. aerogenes bacterium known to be totally avirulent [47]. Other mesophilic A. salmonicida

strains do not possess a complete T3SS. It is the case, among others, of the strains Y567 and Y47

isolated in a food market in India [10] (Table 1). These two strains have also displayed a clear

virulence against D. discoideum (Figure 3).

M18076-11

NBRC 13784T

01-B526

ECFood+05

JF2480

34melT

2004-05MF26

CIP 103209T

JF4097

75 85 95

pectinolytica

masoucida

achromogenes

smithia

salmonicida

0.97 0.98 0.99 1

Figure 2. Cladogram showing the phylogenetic relations between JF2480 and other A. salmonicidastrains. Only bootstrap values inferior to 100 are shown at the corresponding nodes. The heatmaprepresents the ANI values (in the middle) and the POCP values (on the right).

Given the unusual host, the genome sequence was an opportunity to investigate the virulencefactors of the strain JF2480. Secretion systems are protein machineries important for bacterialvirulence [45]. The genome of strain JF2480 was predicted to possess mandatory genes for T1SS, T2SS,T4P, Tad, and T6SSi. Although no core genes encoding for T3SS have been detected, two genes codingfor effectors (AexT and AexU), known to be secreted by this system, were found.

In the past, it has been shown that bacteria without a functional T3SS and especially Aeromonasbacteria were not virulent when tested in the predation assay involving the soil amoeba D. discoideumused as a surrogate host [36,46]. A low quantity of amoebae was able to create a phagocytic plaqueon K. aerogenes (the positive control) while only a high quantity of D. discoideum cells was required toobserve amoebal growth on JF2480 (Figure 3). Thus, bacterial virulence assays conducted on JF2480revealed that this strain is virulent when facing D. discoideum even in absence of a complete T3SScompared to K. aerogenes bacterium known to be totally avirulent [47]. Other mesophilic A. salmonicidastrains do not possess a complete T3SS. It is the case, among others, of the strains Y567 and Y47 isolatedin a food market in India [10] (Table 1). These two strains have also displayed a clear virulence againstD. discoideum (Figure 3).

Figure 3. Virulence of strain JF2480 at low temperature. Different numbers of D. discoideum cells were

deposited on a lawn of the different bacterial strains and grown on HL5 agar for 7 days at 18 °C. This

test has been performed three times (biological replicates) giving similar results each time.

A previous study that investigated the virulence of A. salmonicida from human clinical cases

reported five genes uniquely found in mesophilic strains able to cause necrotizing fasciitis [18]. Two

of these genes, encoding for a hemerythrin and a catalase, were confidently found in the genomes of

JF2480, Y567, and Y47, which are virulent against D. discoideum (Table 2). Homologs of these two

genes were already listed to be involved in the virulence of other bacteria [48,49].

Given the novelty of the host, it was relevant to investigate the genes unique to JF2480 to have

clues about the genetic determinants that may be involved in its success of colonization. A total of 70

genes were found to be unique to JF2480 (Table S3). While several of them encode hypothetical

proteins, others are related to mobile elements (phages and plasmids) and citrate metabolism. A gene

encoding for a zonula occludens toxin-like (Zot) was also found. However, the gene is truncated in

JF2480. By investigating the presence of this gene in other species of the genus Aeromonas, it has been

possible to find that only Aeromonas rivuli has a homologue (Figure S1). The Zot toxin is known to be

a virulence factor encoded in the genome of the lysogenic-filamentous phage CTXΦ that is integrated

in the genome of the human pathogen Vibrio cholerae [50]. The phage CTXΦ also harbors genes

producing the cholera toxin (ctxA and ctxB). These genes do not have clear homologs in the genome

of JF2480, comparatively to the Zot toxin. Further research by performing TBLASTN analysis against

a dataset containing representative genome sequences from all available Aeromonas species (Table

S1), revealed a second gene encoding a putative Zot toxin, which this time seems complete. The

distribution of this gene among Aeromonas is much more scattered than for the partial gene (Figure

S1). For example, several mesophilic and psychrophilic strains of A. salmonicida possess an

homologue of this gene.

Table 2. Presence of the five genes putatively involved in necrotizing fasciitis.

Protein Virulence Trait Strainsc

JF2480 Y567 Y47

Two pore domain

potassium channel family

protein

N/A a 40% d 99% 99%

Hemerythrin A. hydrophila survival in host

macrophages 98% 98% 98%

Pseudaminic acid

cytidylyltransferase Colonisation of H. pylori 99% 50%d 100%

Catalase KatE b Virulence of Leptospira spp. in animal

models 98% 99% 98%

Figure 3. Virulence of strain JF2480 at low temperature. Different numbers of D. discoideum cells weredeposited on a lawn of the different bacterial strains and grown on HL5 agar for 7 days at 18 ◦C.This test has been performed three times (biological replicates) giving similar results each time.

A previous study that investigated the virulence of A. salmonicida from human clinical casesreported five genes uniquely found in mesophilic strains able to cause necrotizing fasciitis [18]. Two ofthese genes, encoding for a hemerythrin and a catalase, were confidently found in the genomes ofJF2480, Y567, and Y47, which are virulent against D. discoideum (Table 2). Homologs of these two geneswere already listed to be involved in the virulence of other bacteria [48,49].

Table 2. Presence of the five genes putatively involved in necrotizing fasciitis.

Protein Virulence TraitStrainsc

JF2480 Y567 Y47

Two pore domain potassiumchannel family protein N/A a 40% d 99% 99%

Hemerythrin A. hydrophila survival in hostmacrophages 98% 98% 98%

Pseudaminic acidcytidylyltransferase Colonisation of H. pylori 99% 50%d 100%

Catalase KatE b Virulence of Leptospira spp. inanimal models 98% 99% 98%

UDP-N-acetylglucosamine-1-phosphate transferase c

Production of enterobacterialantigen in S. enterica 45% d Absent 61% d

a: N/A, none-applicable; b: The catalase was annotated as KatE by PATRIC [33]; c: % of similarity in regard to thevirulent strain A. salmonicida 947 C; d: likely distant homologs.

Given the novelty of the host, it was relevant to investigate the genes unique to JF2480 to haveclues about the genetic determinants that may be involved in its success of colonization. A total of70 genes were found to be unique to JF2480 (Table S3). While several of them encode hypotheticalproteins, others are related to mobile elements (phages and plasmids) and citrate metabolism. A geneencoding for a zonula occludens toxin-like (Zot) was also found. However, the gene is truncated inJF2480. By investigating the presence of this gene in other species of the genus Aeromonas, it has beenpossible to find that only Aeromonas rivuli has a homologue (Figure S1). The Zot toxin is known to be avirulence factor encoded in the genome of the lysogenic-filamentous phage CTXΦ that is integrated inthe genome of the human pathogen Vibrio cholerae [50]. The phage CTXΦ also harbors genes producingthe cholera toxin (ctxA and ctxB). These genes do not have clear homologs in the genome of JF2480,comparatively to the Zot toxin. Further research by performing TBLASTN analysis against a datasetcontaining representative genome sequences from all available Aeromonas species (Table S1), revealed asecond gene encoding a putative Zot toxin, which this time seems complete. The distribution of thisgene among Aeromonas is much more scattered than for the partial gene (Figure S1). For example,several mesophilic and psychrophilic strains of A. salmonicida possess an homologue of this gene.

The amoeba predation assay has been performed at 18 ◦C and not 37 ◦C because D. discoideumcannot survive over 26 ◦C. The virulence of strain JF2480 at this temperature suggests that the bacteriumcan grow quite well at low temperature. To confirm it, the growth curve of strain JF2480, as well asother A. salmonicida strains, was determined (Figure 4). It appears that JF2480 grew as well as theother strains tested at 18 ◦C. In addition, the strain JF2480 is the one with the best growth at 40 ◦Ccompared to other A. salmonicida mesophilic strains (Figure 4A), but this is not statistically significative(p = 1.0000 when comparing JF2480 with masoucida ATCC27013 for example). The difference in growthcapacity is more pronounced at 41 ◦C, with strain JF2480 having the best growth compared to all otherstrains tested (Figure 4B). In this case, it is statistically significant (p < 0.0001 when comparing JF2480with Y567 or Y47 which are the second-best growers). It is interesting to note that the strain from thepectinolytica subspecies showed good growth at 18 ◦C and 40 ◦C, but was much less important at 41 ◦C(p < 0.0001 when compared to A527). The other strains exhibited less fluctuating growth rate variationsat the different temperatures. The growth of the A. salmonicida subspecies masoucida strain was one ofthe lowest at both 40 ◦C and 41 ◦C, which is coherent with previous results suggesting an intermediatelifestyle of this subspecies between psychrophilic and mesophilic [10].Microorganisms 2019, 7, x FOR PEER REVIEW 8 of 13

Figure 4. Growth curves for various A. salmonicida strains. The growth curves were determined at 40

°C (A), 41 °C (B) and 18 °C (C) by measuring the optical density at 595 nm. The means of two (A) and

three biological replicates (B and C) are shown for each strain. Each experiment was also performed

in three technical replicates.

4. Discussion

Historically, the bacterium A. salmonicida was known to infect salmonids, hence the origin of its

name meaning “salmon killer” [51]. Since then, other bacteria belonging to A. salmonicida have been

isolated from various fish species. In order to refine the taxonomy and respond to this diversity,

different subspecies have been defined: salmonicida, smithia, masoucida, and achromogenes. The last

subspecies to be defined, pectinolytica, made it possible to integrate into the salmonicida species an

environmental and mesophilic bacterium, which can grow at a temperature of 37 °C, compared to

0 3 6 9 12

0 4 8 12 16 20 24

Optical density

A527 Y47

JF2480

masoucida ATCC27013

pectinolytica 34melT

Time (h)

A (40°C)

B (41°C)

C (18°C)

Figure 4. Growth curves for various A. salmonicida strains. The growth curves were determined at40 ◦C (A), 41 ◦C (B) and 18 ◦C (C) by measuring the optical density at 595 nm. The means of two(A) and three biological replicates (B and C) are shown for each strain. Each experiment was alsoperformed in three technical replicates.

Enzyme activities and details of the carbon sources utilized by the strain JF2480 are given inTables S4 and S5. Extensive phenotypic characterization tests have been conducted demonstrating thatstrain JF2480 has a more diversified metabolism at 37 ◦C than 25 ◦C with additional carbon sources(D-manitol, D-glucose, palatinose, and D-sorbitol) that can be metabolized. However, even at 25 ◦C,the bacterium presented a wide range of metabolic activities.

4. Discussion

Historically, the bacterium A. salmonicida was known to infect salmonids, hence the origin ofits name meaning “salmon killer” [51]. Since then, other bacteria belonging to A. salmonicida havebeen isolated from various fish species. In order to refine the taxonomy and respond to this diversity,different subspecies have been defined: salmonicida, smithia, masoucida, and achromogenes. The lastsubspecies to be defined, pectinolytica, made it possible to integrate into the salmonicida species anenvironmental and mesophilic bacterium, which can grow at a temperature of 37 ◦C, compared toother subspecies that are restricted to lower temperatures [4]. In recent years, several other mesophilicstrains of A. salmonicida from the environment in India [10,11], and even human patients in Spain andSwitzerland [14,18], have been isolated and characterized at the genomic level.

In this paper, a mesophilic strain of A. salmonicida isolated from a bird that died from septicemia,has been described and analyzed. The discovery of this strain shows that although the different strainsare close evolutionarily (Figure 2), they have a wide range of hosts. The fact that psychrophilic strainsare restricted to temperatures below 25 ◦C considerably limits the hosts that can be infected by themand are therefore specialized in different cold water fish. Mesophilic strains can grow at 37 ◦C (and evenmore, as shown with this study), and also at temperatures as low as those psychrophilic. This suggeststhat mesophilic strains have the potential to infect a wide range of hosts. This idea is supported by aprevious study showing that mesophilic strains recovered from ill humans can also cause infectionin mice [18]. Although strain JF2480 can be considered a pathogenic to Recurvirostra avocetta sinceit was abundantly present in 3 organs of a freshly dead bird, we do not know if the JF2480 strainis a polyvalent pathogen or if it is also, like the psychrophilic strains, specialized to specific hosts.On the other hand, the capacity of the JF2480 strain and other mesophilic strains to resist D. discoideumpredation, a psychrophilic protozoan, opens the door to a wide range of hosts for mesophilic strains.Further infection experiments using mesophilic strains on fish will be mandatory in the future to helpanswer this question.

One of the reasons why the hosts that may be infected by mesophilic strains are still cryptic isthat no strain has yet been isolated twice, thus making it impossible to determine confidently thereal host range. Until very recently, A. salmonicida was mostly known as a pathogen of cold-waterfish. It is therefore realistic to think that infections caused by these bacteria in other hosts havebeen falsely under-diagnosed. For example, we know that A. salmonicida can easily be diagnosed asA. hydrophila and that molecular analyses are needed to confirm the taxonomic identity of the strainswith certainty [9,18].

In addition to demystifying their host range, the isolation of other mesophilic strains ofA. salmonicida will shed light on their pathogenicity. As discussed elsewhere, T3SS is known tobe a virulence factor essential to the pathogenicity of strains of the salmonicida subspecies [52].Although T3SS seems to also be important for virulence of mesophilic strains [18], it is likely notessential as some virulent strains, as JF2480 described in the present study, lack it. Even withouthaving a T3SS, JF2480 bears two effectors related to this system: AexT, a binary toxin with actinADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42 [53] and AexU. It is unclear ifthese effectors are the remnant of a previous T3SS or if they can be translocated by another secretionsystem. In A. salmonicida subsp. salmonicida, the T3SS locus is known to be located on a thermosensitiveregion that can be lost when strains are grown at a high temperature (>25 ◦C) [45]. It is still unclear if aformer T3SS in JF2480 may have been lost in such a way.

As previously suggested [18], and reinforced by the present study, other genes also seem to beimportant for the virulence of mesophilic strains. These included five genes known to be virulencefactors in other bacteria and potentially involved in necrotizing fasciitis by the mesophilic strainsof A. salmonicida (Table 2). In the present study, we found two genes encoding zonula occludenstoxin-like (Zot) in the genome of JF2480 (one partial and one likely complete), which is a virulencefactor in V. cholerae [50]. In V. cholerae, Zot is encoded by the lysogenic phage CTXΦ along with thegenes producing the cholera toxin. In the case of strain JF2480, the genes encoding Zot toxins are notpredicted to be encoded on a mobile DNA element such as a prophage. In addition, genes encodingcholera toxin do not have homologues in JF2480. The fact that the partial gene is found at the momentonly in strain JF2480 among A. salmonicida, suggests a recent acquisition horizontally, and not a verticaltransfer. This is interesting in the context where it has been shown that the strains of the psychrophilicsubspecies A. salmonicida subsp. salmonicida can acquire exogenous DNA from other pathogens, suchas Salmonella enterica [42,43] and Chlamydia suis [44]. The present study suggests that some mesophilicstrains of A. salmonicida may also adapt by acquiring genetic material from other pathogens andthus play a role in the dispersal of these genes. Regarding the likely complete gene, given its broaddistribution among Aeromonas species (including other strains of A. salmonicida), it is likely not solelyresponsible for the bird infection, although it might have contributed as a virulence factor.

Several genes involved in citrate metabolism were also found. The use of citrate was already shownto be important for virulence of pathogens such as Mycobacterium tuberculosis, Staphylococcus aureus,and V. cholerae [54–56]. Although there is currently no evidence of such involvement in JF2480, thismay be a clue to help decipher why these genes are present in this strain.

5. Conclusions

In conclusion, the discovery and description of the JF2480 strain, the first known A. salmonicidastrain isolated from a bird, has become further support that this species can infect a wider array ofhosts than previously suspected, and that we need to rethink the way we perceive A. salmonicida’snatural environment. In addition, the JF2480 strain is additional proof that the mesophilic groupof the salmonicida species is more complex and diversified than the psychrophilic one. As alreadyproposed [11,18], it is mandatory to continue this effort to isolate and describe new mesophilicA. salmonicida strains from different sources and hosts to finally have a global portrait of thisbacterial species.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/7/12/592/s1,Table S1. Genome sequences of Aeromonas used for the phylogenetic analysis; Table S2. Best-fit model of eachpartition; Table S3. Genes found to be unique to JF2480; Table S4. Phenotypic characterization of A. salmonicidastrain JF2480 at 37 ◦C using a VITEK 2 system from bioMerieux; Table S5. Phenotypic characteristics of A. salmonicidastrain JF2480 using a API bioMerieux apparatus; Figure S1. Distribution of the genes encoding the Zot toxin in thegenus Aeromonas and percentage of similarity when present.

Author Contributions: A.T.V., J.F. and S.J.C. contributed to the concepts and design of the study. A.T.V., A.B., J.F.and S.J.C. participated in the acquisition, analysis, and interpretation of the data. S.J.C., A.T.V. and J.F. drafted themanuscript. All authors reviewed the manuscript.

Funding: This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).ATV received a Postdoctoral Fellowship from NSERC. SJC is a research scholar of the Fonds de Recherche duQuébec en Santé.

Conflicts of Interest: The authors declare no conflict of interest.

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